The diagnosis of bee diseases by laboratory methods

Hee R ibe si yh \\ ts
uhh Vig} { { ie a Nh

AAAS AN
|
ih?

a

PLAC AGM MECC HH Ra MR Teapot
’ Vi 41 3 HAE Hh ny \ iy Wn Ath iui “ ae

i} th a ie : a panies

14) eave Lae bee nit Yih it A _ vet a

al tuk te it *

"i i au i ita ite of ys wi _ : . an r “a a whe a i i

f CO ae mh aah ai ang en ry egbaceR th aa
b Pah Ri ‘ut Wit a mi ice area ahy-4 : . ‘i
‘ ANT 1a vie ity i a ae ae be nat

any a Nt
i 4) Hin a

+)!

————

Ai \ Hike Rye ie dae
AEPSRGr tibet is RiP ATL
AeA Ente rebel qi Sanhiy Di AGN \

{ MARRY

poe Hy

‘ es i Haiti ry h,
ae oo ce
rte ett

it
is Sinan . "i cir
ae i we tt thet a sh
sit at i i ioe
i. st) se a in vhs sa ay
Lb; de Pap be tt bet
i‘ wih ina fie it as iy
Meals Hie RUA Uae Mpa

eee fen
Se =
~<,

mbt Grete Set EAE, st) i a
. ae
tf \
ee ‘3 be Hy

ut ( ie)
pai haast ne
ety

eur
AR

oe
M4 }

ae ee ee
Pret oreen A >
ao eee = Sar ‘ Set
<

oT Wal GE wy aye

Up Apres peut beaks Be iy f +f

Hitt alive iis ena AEH BN ht Pas tie

Hy Bi Pa EUS yet Sine

oye pH iy SNORE
i ae Ob

=

ee = cis aes — SS = — pases pee Zee E erate =

wee == = ere = ere eee ee = apa aay - me = 3 eceseate =
Ste sia ee oe aes a ~ et Sat ake ——- : : Sass 2 =

: a

Uw pen, 7 s,
ae Jae “s .

7:

ar ETRE eS
Sec ery
re eS

Se ee

hoe aN
: ; oo
Hats Sit a Ents

Ane aby
ok: tee)

ote eee
Ree tes

i

ic
<2
eS

Ste:

iY

Rete ee as

eeaceaaes
ay e eae =

Se
se

ya Barat

Se ASR Se

< 7 ?

pees,
Fae: Ce

Seen
a Pet oa
See eas
eyes Tay
‘e Fars
eee
S32
ers

~

a eer

faba bao

ares: 3

Eis =
==.
Res
s
~~

tne tin

>
este:
a
==
Set n=¢
ae qoeste
Pa Satne,
ore
rote

Let

Pee.
Sys

3 eee &
—* eee :
a aan
as
=F

~s
CES

a

3.

2 FB yA.
2 eas ~"
SRE OE rs aed ea ee Ss
Saye ote PhS Re oe
a ~ YY a Ie
tees
ae Sete

segs. =
ee oe

~
mt

Se

aH + Mii:
i a
iat +e nik re j if
I 1H) ¢ i he
bai a
¢ K 3a)

este es
=

aoa
5 EN

passes

at eee
aS
ms
Saree

pees
f<Sars
Sate
Rim ecu!
7.

ne
cee

er Cate

» —S
Spee eee
Sere
; : <
oe
=)

I -
Secaeeeae

Sepa an

ae
toe

—

Se ee
or

At

Age:
jeiataas ae : nad Peep
ty TAT gD {Wal otek : q Yi RAM? has ; acess
Ba yh sats are \ ne HR MnO canal aa a et aa ce oe ae
. 5 il Bik : ‘ ey ty yeah lagen, Die er ba ces ty : ay ied i j 3 a) j
, POM MANNED ei ip ret a i aid fa ntact Leia ait pet Littw ana oe Hare Ro
MEHR ICRC (otaiB trate rtatege th fute(: Nake Ce EAC ets E sie at cee
tae )oPOTE DDE Tada aabaad ahead esate fet tet te tlt

cee
ens

Historic, archived document

Do not assume content reflects current
scientific knowledge, policies, or practices.

oy

}
fan
k
Le

n

UNITED STATES DEPARTMENT OF AGRICULTURE

| BULLETIN No. 671 §

a
Di
WAS
= +.
“\ TBI)

Contribution from the Bureau of Entomology
L. O. HOWARD, Chief

Washington, D. C. PROFESSIONAL PAPER. June 21, 1918

THE DIAGNOSIS OF BEE DISEASES BY
LABORATORY METHODS.

By ArtHur H. McCray,
Apicultural Assistant,
and
| G. F. WHITE,
Expert Engaged in the Investigation of Bee Diseases.

CONTENTS.
Page Page
DEMMGLICHIOU Eee een rey Pa es 1 | Diseases to be diagnosed—Continued.
Poporatory moeunods ses 4.25... 3.0.25. kee 2 Sacbroodl Jos esse ieee wees 10
Diseases to be diagnosed.................--- 4 Other abnormal conditions of the brood. 11
European foulbrood...-............-..-- a INoSemardiseasele tas 2 aka eee es ee 13
American foulbrood |. 2-224. Ses 8 Miterature- sett ti 2c eee ee hea ele 14
INTRODUCTION.

Already some of the States have established laboratories for the
diagnosis and investigation of the diseases of bees. The number of
State laboratories where a rapid and accurate diagnosis of suspected
material can be made will no doubt increase in the future in pro-
portion as State laws for bee-disease inspection and control are insti-
tuted and perfected. Inspectors realize that there will be always
some suspected brood and many conditions among adult bees which
can not be diagnosed in the field, but which will require laboratory
_ methods for diagnosis. In addition to the demands made by in-
_ spectors for the examination of suspected material, there will be
numerous independent requests from beekeepers.

During the past several years the writers have examined a large
number of specimens of suspected brood and bees sent to the Bureau
of Entomology, and have developed and perfected methods and
technique in the diagnosis of the known diseases of bees which, it is
believed, will prove valuable to others. It is the aim of this paper
_ to present these methods of diagnosis for the benefit of those who
may engage in similar work.

47164°—Bul. 671181

2 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE.

For the understanding and application of the methods herein out-
lined, a preliminary training in general bacteriology, supplemented
by a special knowledge of the pathogenic bacteria and the methods
pursued in the diagnosis of diseases in general, is essential. As effi-
ciency depends largely upon a knowledge of pathology, too much
emphasis can not be given to it. With such general and special train-
ing as a basis, and with a knowledge of the bee diseases, the acquisi-
tion of the special methods necessary for a laboratory diagnosis of
them becomes a comparatively simple matter.

One of the authors (White) began his investigations on the dis-
eases of bees in 1902, and from this date to 1909 examined and
diagnosed about 500 samples of suspected material and during the
period developed the laboratory methods as given in the present
paper. Since 1909 the senior author (McCray) has carried on the
work of diagnosing the samples received by the Bureau of Ento-
mology and up to the present time has examined and diagnosed
about 5,000 of them.

LABORATORY METHODS.

OBTAINING THE SAMPLES.

For a satisfactory diagnosis of suspected brood the material at
hand should be adequate. The size of the comb containing the af-
fected larvee should be ample. <A piece about 5 inches square is suit~
able, provided it contains a sufficient number of larve. All samples,
no matter how far sent or how long in transit, should be inclosed in
wooden boxes to prevent crushing during transportation. Paste-
board boxes are apt to allow the sample to be crushed, while samples
inclosed in tin boxes are often covered with a rich fungous growth
when received. Both the crushing of the sample and the fungous
growth obscure the gross features which form an important part of
the examination. The sample should not be wrapped in cloth or
paper, as this will tend to retain moisture and further encourage
fungous growth. If thin paper is used, it frequently adheres so
closely that its complete removal is difficult. The sample should con-
tain no honey, as a small amount of it in a comb is sometimes sufii-
cient to cause an unsightly mass, owing to leakage through the pack-
age in transit. As complete a history as can be obtained should
accompany each suspected specimen as an aid to diagnosis. The
Bureau of Entomology for several years past has sent out a printed
question card with blank spaces for answers by the beekeeper sending
the suspected brood. Some of the more important of these questions
are as follows: |

1. Is there any bee disease in your neighborhood?

2. If so, what disease?

3. Have you brought colonies from a distance? If so, give source.

DIAGNOSIS OF BEE DISEASES. 3

4. Have you fed honey from other sources than your own apiary? Give source
if known.
. Is the diseased brood mostly capped or is it mostly uncapped?
. What is the color of the larve (grubs) soon after death?
. Later, what is the color of the decaying larve?
. Are the dead larval remains ropy?

. Do you notice any disagreeable odor in the hive?

10. Does there seem to be an unusual number of queenless colonies in the
apiary?

11. What disease do you suspect?

12. Give location of the apiary from which sample was taken by town (or
township) and county.

OMOnID

While a diagnosis sometimes can be made from larve which have
been removed from their cells, and sent without the comb, such mate-
rial is not satisfactory. It is far better to examine the infected larve
in the comb in which they die. The diagnosis of bee diseases should
not be based upon the examination of honey alone.

EXAMINATION OF THE SAMPLES.

In diagnosis both gross and microscopic examinations are made of
the suspected material.

GROSS EXAMINATION.

The following points are to be taken into consideration: Character
of the caps; regularity of the brood; proportion of affected brood;
position of diseased larve within the cell; age, color, consistency, and

odor of the affected brood; and kind of larvee affected, whether queen,
drone, or worker brood. These factors will be taken up in detail
as each brood disease is considered separately.

In the gross examination of the comb it should be held in such
a manner that a good lighting of the interior of the cells is secured.
This is especially important in examining for scales. The best
method of examining suspected brood is to hold the comb in a ver-
tical position and about level with the eyes; then, by gradually in-
-clining the top of the comb toward the observer, a point is reached
at which the greatest amount of light is thrown upon the floor of the
cells. This brings out the scales with great prominence and permits
of their close scrutiny within the cells. In examining the affected
brood for consistency and adherence to the cell walls in the scale
‘stage, a small pair of curved forceps is convenient.

The gross examination of the adult bees will be considered under
Nosema disease.

In routine diagnostic work unstained water mounts have been
found very satisfactory in searching for spores, and stained prepara-
tions are made for the vegetative forms. Carbol fuchsin is a suitable
stain to use.

4. BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE.

MICROSCOPIC EXAMINATION.

The number of larve or pupz to be examined in a given sample
depends upon various factors. If the case is a typical one, one larva
or pupa dead of a disease usually is sufficient. If, on the other hand,
the gross appearance is not so definite and the microscopic picture
from the first dead remains examined is unsatisfactory, others must
be studied. While much might be written concerning the microscopic
appearance of smears from larve or pupe affected with the various
brood diseases, as compared each with the others and with smears
from healthy brood, such elaborate descriptions are not deemed ad-
visable in this paper. Only the more salient features of the micro-
scopic picture will be given for each disease considered, as it is
believed that such descriptions will best serve those for whose benefit
the paper is prepared.

It will be understood that when the authors write of the recog-
nition of certain organisms by microscopic examination, as, for
example, that of Bacillus pluton or Bacillus alvei, either in stained
smears made from tissue or in stained smears made from agar plates,
they refer to the recognition of the organism under observation only
in a general way, meaning rather that the microscopic picture sug-
gests the organism. The identification of the organism is complete,
naturally, only after a consideration of its cultural characteristics
also.

CULTURES.

In culturing the affected brood agar as ordinarily prepared in the

laboratory is used in making plates. Those larve or pupe are

selected which upon microscopic examination have shown evidence ©

of disease. Of course as many additional ones may be cultured as
desired. The cultures are incubated for different pericds of time, as
will be noted in the discussion of the diseases.

Dead, not living, larve are examined.—In the laboratory examina- ~

tion of diseased bee brood, the affected larve are always received
dead, therefore nothing will be said relative to symptoms and appear-
ance of affected living larve.

DISEASES TO BE DIAGNOSED.

EUROPEAN FOULBROOD. A

European foulbrood is an infectious disease of the brood of bees”

caused by Bacillus pluton (White, 1912).
GROSS CHARACTERS.

(a) The caps and regularity of the brood —tUarve that die of
European foulbrood do so usually before they reach the age at
which brood is capped. Brood dead of the disease is therefore usu-
ally found in uncapped cells. When the larve die after capping

’
f

DIAGNOSIS OF BEE DISEASES. 5

the caps usually are entire, but may be punctured. The caps may be
slightly sunken, but usually are not. Owing to the fact that such
a small percentage of the affected brood becomes sealed, a comb of
brood affected with European foulbrood with its few sealed cells and
Jarge amount of young uncapped brood presents a distinctive appear-
ance (PI. I, fig. 1) and shows a marked contrast to the solid areas
of brood of uniform age in healthy combs.

(6) Proportion of affected brood—Kuropean foulbrood usually
has made rather extensive ravages by the time the beekeeper detects
it; hence in many samples received for diagnosis a very large pro-
portion of the larve in the comb are affected. Toward autumn,
however, it is not unusual to receive samples containing a small num-
ber of affected larvee.

(ce) Position of larve within the cell—The usual position for
larvee affected with European foulbrood is that of lying curled at the
bottom of the cell. Other affected larvee lie extended in the cell,
but these are few in number.

(d) Age of the dead larve—tIn most of the specimens received
for diagnosis by far the larger proportion of the affected larve are
young, lying curled at the bottom of the cell as just stated. Besides
this comparatively young brood, older larvee, including a few sealed
ones, may be found affected.

(e) Color—A change in color is one of the first abnormalities
noted in brood dead of European foulbrood. Yellow or gray and
combinations of these two colors are among the first to be noted.
Later the yellow and gray gradually deepen, until quite a dark brown
is attained. Larve dead of this disease often present a peculiar
appearance, as though they were melting away under the influence of
heat. The transverse tracheal branches stand out prominently. This
melting appearance of the larvee, the yellow, gray, and brown colora-
tion, prominent tracheal branches, and large amount of uncapped
affected brood are characteristics not easily confused, in the majority
of cases, with those of other diseased conditions of the brood.

(7) Consistency—tLarve dead of European. foulbrood are com-
paratively friable. However, larvae which are somewhat viscid usu-
ally may be found. Perhaps slimy, rather than viscid, expresses
better the consistency of some of these larve. ,

(g) Odor.—A slight, inoffensive odor is frequently to be noted in
European foulbrood. The yeastlike odor which has been described
is not constant in brood affected with European foulbrood. A simi-
lar odor may be detected in samples other than those which contain
European foulbrood.

(h) Kind of brood affected—Sometimes samples are received
which contain only affected drone-brood. Most cases, however, con-
sist only of worker-brood. Qucen larve also may be attacked.

6 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE.

(7) Scales—Scales are formed by the drying of the affected
larve, and from the foregoing description of the dead brood some
conception may be gained as to their form and appearance. Scales
of European foulbrood, like most of the affected larvee, are small and
lie at the bottom of the cell, from which they can be separated with
ease. The color of the scale is in general yeilow, gray, or brown,
and the cross markings formed by the transverse tracheal branches
usually are still in evidence. This is the usual type of scale found
in European foulbrood. Occasionally there will be received, how-
ever, a sample containing only a few scales, or perhaps a single
scale, in marked contrast to the scale just described. These scales
are always few in number in a given comb area, are usually dark
brown in color, are less easily removed than the small ones, and are
not brittle but rubberlike in consistency. |

MICROSCOPIC FINDINGS.

The appearance of Bacillus pluton, the etiological factor in Eu-
ropean foulbrood, in stained preparations usually is sufficiently char-
acteristic to render its microscopic identification comparatively cer-
tain. Besides Bacillus pluton, the following secondary invaders
may be found: Bacillus alvei, Streptococcus apis, Bacillus vulgatus,
Bacillus mesentericus, Bacillus orpheus, and Bacterium eurydice.
Without careful observation Bacillus pluton and Streptococcus apis
might be confused. Upon careful examination it is found that
Bacillus pluton presents considerable variation in size and morphol-
ogy in the individual organisms. Some of them occur in the form
of cocci, yet the general picture is that of an organism with more
or less pointed ends. Thin smears should be made in order to obtain
details of morphology.

It is important to have a true conception of the microscopic appear-
ance of Bacillus pluton. 'The essential facts are the typical mor-
phology and the manner of grouping of the individual organisms.
The general shape of the group is often more or less circular, although
numerous groups of more or less irregular form may be observed.
Groups of varying shapes and sizes will be noted as successive fields
are brought into view. It is the presence of these groups, containing
a sufficient number of organisms with the pointed ends described,
that serves to differentiate Bacillus pluton from Streptococcus apis.
Streptococcus apis usually occurs in forms which are sufficiently
coccuslike to lead to little or no hesitancy in differentiating it from —
Bacillus pluton. Forms which are sufficiently pointed to resemble
B. pluton do occur, however, and if only a few are present in a field
the differentiation of these species is not possible. By making a
sufficient number of smears from a sufficient number of larvee, forms

DIAGNOSIS OF BEE DISEASES. Tf

_ In abundance typical of either 2. pluton or Strep. apis usually can be
found. In stained smears of Strep. apis the organisms are found to
be spread out over the whole field with no tendency to grouping as in
* the case of B. pluton. Sometimes in examining European foulbrood
larve the microscopic picture shows practically nothing but B. pluton.
More often, however, Strep. apis, B. alvei, and other rod forms are
found. Some larve will disclose B. alvei alone, others Strep. apis
alone, and still others, these two organisms without B. pluton. Con-
tinued search is sometimes necessary before larve are found reveal-
ing B. pluton, either alone or with one or more of the secondary
invaders just mentioned. The authors have found Bacillus pluton in
the small yellow, gray, and brown scales as well as in the soft melting
larve. They are not prepared, however, to state the length of time
that the organism persists in the dried state. Mention has been
made of larger scales of rubberlike consistency which occur only
occasionally and in small numbers in a given comb. Such scales
always yield microscopically Bacillus alvei in abundance, and usually
this organism alone. The microscopic appearance of B. alvei in the
spore stage is rather characteristic, the spores practically always
showing vestiges of the rods clinging to them. This aids in dif-
ferentiating it from B. vulgatus and B. mesentericus. Bacillus
orpheus may be recognized microscopically in the spore stage by
the position of the spore in the rod, it being eccentrically placed.
Bacterium eurydice is a small, slender organism which does not
form spores.
CULTURES.

At the present writing no medium suitable for growing Bacillus
pluton has been devised, hence agar plates made from European
foulbrood larvee show only the secondary invaders—B. alvei, Strep.
apis, B. vulgatus, B. mesentericus, and B. orpheus. Bacillus alvei is
encountered very frequently and is always secured on culturing larvee
in which the microscopic examination has revealed the presence of the
organism. Streptococcus apis occurs occasionally. Bacillus vulga-
tus and B. mesentericus frequently are met, but usually in small
numbers only. Bacillus orpheus in large numbers is occasionally
encountered. Bacterium eurydice, as a rule, does not appear in the
cultures. Bacillus alvei is the only organism occurring with any
marked degree of frequency and in any great numbers on agar
plates made from affected larve of any of the known infectious brood
diseases of bees. Rarely do cultures from larve dead from any
cause other than European foulbrood show the presence of this
species.

The appearance of B. alvei on agar plates is rather characteristic.
The colonies usually occur in abundance, often being innumerable.

8 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE.

When a few colonies are present there is seen, under low magnifica-
tion, a granular center for each colony surrounded by numerous
smaller but similar growths. There is little chance for error in the
identification of B. alvez, assuming that the gross characters of the
suspected material cultured had suggested European foulbrood and
that the microscopic examination of the material had suggested the
organism.

Cultures should be incubated until the second day in making a
diagnosis of suspected European foulbrood material, since spores of
B. alvei are not produced in abundance until that time. Two days,
then, is the minimum time in which a report can be rendered on this
disease. B. vulgatus, B. mesentericus, and B. orpheus may be recog-
nized, when present, by their morphology and cultural character-
istics (McCray, 1917).

AMERICAN FOULBROOD.

American foulbrood is an infectious disease of the brood of bees
caused by Bacillus larvae (White, 1907).

GROSS CHARACTERS.

(a) The caps and regularity of the brood.—A \arge amount of the
affected brood is capped, and many of the caps may be sunken and
many perforated. The coloration, the sunken and perforated caps,
and the irregularity produced by the capped and uncapped cells
present quite a characteristic appearance (PI. I, fig. 2).

(6) Proportion of affected brood—vThe proportion of affected to
healthy brood in American foulbrood is, as a rule, high, although
specimens secured early in the attack may show a considerable pro-
portion of unaffected brood.

(c) Position of the larve within the cell—Inasmuch as most of
the larve in American foulbrood die after the time of capping, the
position of the larve is that of extension along the floor of the cell.
But the dead larve quickly lose their form and symmetry, so that a
dark, shapeless mass soon occupies the lower portion and bottom of
the cell.

(d) Age of the affected larve.—The usual age at which the larve
are found dead of American foulbrood is just after the time of seal-
ing. This fact is of importance in considering the size of the scale
and its position within the cell. Rarely is young unsealed brood
found affected in this disease.

(e) Color—Most of the dead larve when received for diagnosis
will be of a dark chocolate color. Only rarely are larve of the lighter
shades of brown seen. The late stages of decay are very dark brown.

(7) Consistency.—The consistency of the affected larve is char-
acteristic and pathognomonic. The larve are strikingly viscid, so

PLATE I.

Bul. 671, U. S. Dept. of Agriculture.

—COMB CONTAINING LARVA DEAD OF EUROPEAN FOULBROOD.

Fia 1

(Original.)

About natural size.

Fic. 2.—COmMB CONTAINING LARVA DEAD OF AMERICAN FOULBROOD.

(Original.)
EUROPEAN AND AMERICAN FOULBROOD

About natural size.

Bul. 671, U S. Dept. of Agriculture. PLATE II.

go!
=
=

> nn at aie = a gas ° * lt — set a Sf
ObaaA Ar eae
Fia. 1.—COMB CONTAINING LARV4 DEAD OF SACBROOD.

N
Natural size. (White.)

se

#0 “gf

CONG s* Mee
y é

,o 4, aK .

Win S258 af 4

LR Fe ONG

a 4 3? 4
Fea
at 8

44
59) Pe, ~~

¢. -s ®% a. ca 4 ~ %
“-? at tne og att
‘ EF et Pe bear 2G 00 OU Se
i? Ol Ve 4 ait one =
re ea oe IG!

Fic. 2.—STAINED SMEAR PREPARATION SHOWING SPORES OF NOSEMA APIS.
Highly magnified. (Original.)

SACBROOD AND NOSEMA SPORES.

DIAGNOSIS OF BEE DISEASES. 9

that on thrusting the forceps into the brown larval remains and
withdrawing them a portion of the decaying mass adheres and is
drawn out, often to a distance of 3 or 4 inches. The viscidity is
often referred to by the term “ropiness” in beekeeping literature.
In the rare instances in which young uncapped affected larve are en-
countered the ropiness is less pronounced.

(¢) Odor—The odor is characteristic and may be described as
unpleasant. Often it is feeble or absent altogether, probably having
disappeared after the removal of the diseased brood from the hive.
Affected brood-comb will absorb other odors if given the oppor-
tunity, thus masking the original characteristic odor. Such dis-
appearance and masking of the odor has been observed where speci-
mens of diseased brood in combs from various sources had been
thrown together and allowed to le about preparatory to being de-
stroyed (McCray, 1916).

(h) Kind of brood.—lIt is affected worker-brood that is most often
encountered in American foulbrood samples, although drone-brood
is sometimes affected.

(z) Scales—tThe scales of American foulbrood are distinctive and
are characteristic of the disease. If they are present in sufficient
numbers the disease can be diagnosed from the gross appearance
alone. The lower cell walls can be easily illumined by tilting the
upper portion of the comb toward the observer, as previously de-
scribed. The scales appear extended along the lower cell wall, are
quite dark in color, and adhere closely to the floor and base of the
cell. Sometimes they adhere so closely as to break when an attempt
is made to remove them from the cell. Often a semblance of the
form of the pup is evident in the dried-down mass forming the
scale. Some of the mouth rarts of the pup sometimes protrude
sufficiently to adhere to the root of the cell.

MICROSCOPIC FINDINGS.

In considering the microscopic appearance of stained smears from
infected brood in this disease, there usually is only Bacillus larvae
in the spore form to engage the attention of the observer. In some
imstances, however, samples containing the disease in its earlier
stages are received and then Bacillus larvae in the rod or vegetative
form may be encountered. Such a sample usually is more difficult
to diagnose. To receive a sample in which some older larve con-
taining spores of Bacillus larvae can not be found, however, is a
very rare occurrence.

In a stained smear made from an infected larva and mounted in
water, the microscopic picture is rather characteristic. The most
striking feature of the mount is the large number of spores adher-
ing to the cover glass, floating with the current, or dancing free in

10 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE.

the water medium. Many of the spores stain slightly about the
periphery, which aids somewhat in the observation. Good results
may be obtained from an unstained water mount. The vegetative
forms of Bacillus larvae, when they are present, are observed to be
slender rods, which tend to occur in chains.

CULTURES.

In culturing affected larvee it is the absence of growth on the agar
plates that is important in the diagnosis of American foulbrood.
This is because the spores of Bacillus larvae will not germinate and
grow on the ordinary media of the laboratory, and other growth is
absent because there are seldom secondary invaders present. Occa-
sionally there will be a spreading growth of B. vulgatus, or B. mesen-
tericus, and very rarely of B. alvei. On quite rare occasions a con-
siderable number of colonies of B. vulgatus or B. mesentericus have
been found. As both of these species form spores, as a rule, within
24 hours, their differentiation from B. alvez usually can be made in
this way. A report on a sample of American foulbrood, therefore,
nearly always can be made within a day.

SACBROOD.

Sacbrood is an infectious brood disease of bees caused by a filter-
able virus (White, 1913 and 1917).

GROSS CHARACTERS.

(a) Character of caps and regularity of the brood.—Uarve usually
die after capping in this disease, some of the dead brood being un-
capped by the bees later. Occasionally the caps are punctured. An
area of comb affected with sacbrood therefore presents an irregularity.
So far as the age of the affected larve themselves is concerned, there
is considerable uniformity owing to the fact that death in this disease
occurs after sealing during the two-day period of rest just preceding
pupation. The affected brood, however, is interspersed among healthy
brood of varying age, which adds to the irregular appearance of the
affected comb (PI. II, fig. 1).

(0) Proportion of affected brood. —As a rule there is not a large
proportion of affected brood in a given comb area. Often there will
be an affected larva only here and there.

(c) Position within the cell_—The position of the affected larve is
that of extension lengthwise along the floor of the cell, against which
the dorsal portion of the larva lies. The head is turned upward, to-
ward the roof of the cell.

(d) Age—The brood dies after it has been sealed.

(e) Color—uUsually by the time brood is received for diagnosis the
color of the affected larve is brown or quite dark—often almost
black. If the brood is in the earlier stages of decay, however, the

DIAGNOSIS OF BEE DISEASES. ah

color may be light yellow, light gray,.or ight brown. The lighter
shades soon deepen to the darker ones.

(7) Conststency.—The consistency is characteristic. The cuticular
portion of the body wall of an affected larva is decidedly resistant so
that the larva may be grasped with forceps and removed from the
cell intact. After removal from the cell the larva has the appear-
ance of a small closed sac. When the sac is ruptured the contents
will be seen to be watery. Suspended in the waterlike fluid will be
noted numerous fine brown granules.

(g) Odor.—There is no distinctive odor to sacbrood combs.

(h) Kind of brood.—The greatest ravages occur in the worker-
brood. Affected drone-brood may be encountered.

(¢) Scales——The scales when dried down are quite black and the
surface appears somewhat roughened. They separate readily from
the cell wall and may be lifted out intact by means of forceps.

MICROSCOPIC FINDINGS.

The striking feature of the microscopic examination is the absence
of microorganisms. Rarely a few rods may be observed. A large
amount of detritus is always in evidence, consisting of the brown
granular material seen on gross examination after rupturing the
body wall of the larva. These granules are in a large part the re-
sult of the disintegration of the fat body of the larva.

CULTURES.

As might be expected from the microscopic examination, agar
plates inoculated with infected material are practically always nega-
tive as to bacterial growth. Even the presence of organisms of the
vulgatus group is rare. No other growth occurs unless from chance
contamination.

OTHER ABNORMAL CONDITIONS OF THE BROOD.

There are noninfectious abnormal conditions of the brood of bees
which have been confused with one or more of the infectious diseases.
’ Among the more important of these may be mentioned chilled brood,
starved brood, overheated brood, drone-brood resulting from laying
workers, and brood dying after removal from the hive. The names
given to most of the foregoing conditions are sufficient to indicate in
a general way the probable cause of death. These conditions are less
likely to be confused with American foulbrood than with either one
of the other two infectious brood diseases of bees. The specimens
that resemble European foulbrood in the gross consist of soft, easily
ruptured, gray, yellow, and light-brown larve. The irregularity of
the brood, the age, the color, and sometimes the scales bear a strik-
ing resemblance to many cases of the disease.

On microscopical examination of the affected larve the smear
often discloses microorganisms, vet they lack the definite, clear-cut,

12 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE.

positive picture desired in the diagnosis of the disease. If, on cultur-
ing, the agar plates are free from Bacillus alvei the specimen is usu-
ally considered negative.

Occasionally specimens are received resembling sacbrood that tend
to cause confusion. The head in some of these has a tendency to turn
upward, resembling sacbrood, but unless there is present the tough
body wall and watery granular contents, a diagnosis of sacbrood
should not be made. A resemblance to the disease is sometimes noted
after the brood dries down to form a scale, dark in color and sepa-
rating readily from the cell wall. The microscopic examination and
the cultures are often negative as in sacbrood. In such cases, when
there are only a few affected larve, it is impossible to make a diag-
nosis. Samples of comb containing only pollen without brood or
scales have been received for diagnosis. Such specimens are always
unsatisfactory and insufficient for diagnosis.

TABLE 1.—Differential features in the diagnosis of the brood diseases of bees by

laboratory methods.

European foulbrood. |
|

American foulbrood.

General appearance of
brood.

Proportion of affected
brood.

Position within cell... -_-

Age of the larve...._..-

Colorationes=s—eae eee

Kind of brood affected..

Sealesiey sso eee Nok ee

Microscopic findings...

Culfurest eee see eee

Usually die before capping...

.| Bacillus

Brood irregular. Large
amount of afiected brood
unsealed.

Varying number of young
ae affected, usually

Usually curled at bottom.
Larve soft, with melting
appearance.

Larve yellow, and

brown.

gray,

Slight, inoffensive

Soft, rather friable..........-

Often considerable amount
of drone-brood as well as
worker-brood.

Usually small and lie at bot-
tomofcell. Yellow, gray,
or brownin color. Some-
times a few larger, brown,
rubberlike scales. All
scales separate readily
from cell wall.

pluton always.
Bacillus alvei usually.
Streptococcus apis some-
times. Bacillus orpheus,
Bacterium eurydice, Bacil-
lus vulgatus, and Bacillus
mesentericus, occasionally.

Any of the above organisms
except Bacillus pluton.

Very irregular; affected
brood sealed, sunken
and perforated caps
present.

Usually a large amount
of brood affected.

Extension along lower
cellwall. Larvz soon
become a _ shapeless
mass.

Usually die after capping

Usually dark chocolate...

Usually strong charac-
teristic odor. More or
less offensive.

Viscid, can be ‘‘roped”’
out a distance of 3 or
4 inches.

Any considerable
amount of drone-brood
less likely to be seen.

Extension along lower
cell wall dark
brown in color. Sur-
faces Somewhat
smooth. Separate
from cell with diffi-
culty.

Usually only Bacillus
larvae. Occasionally
Bacillus vulgatus and
Bacillus mesentericus.

Frequently negative.

Sacbrood.

Brooaless irregular,
perforated caps pres-
ent, dark sunken
caps not so pro-
nounced asin Ameri-
ean foulbrood.

Small amount of brood
affected.

Extension along ioavor
cell wall. Head
turned upward.
Normal form main-
tained.

Almost invariably die

after capping.

Scon become dark
brown to almost
black.

None.

Contents watery and
granular. Laryve can
be removed from cell

‘without rupturing
body wall.

Greatest ravages among
worker-brood.

Extension along lower
cell wall. Dark in
color, often black.
Somewhat rough-
ened appearance.
Separate readily from
cell wall.

Negative as a rule.

| Nearly always wholly

Never Bacillus larvae |

on common media.

|

negative.

DIAGNOSIS OF BEE DISEASES. en

NOSEMA DISEASE.

Nosema disease is an infectious disease of adult bees. It is the only
adult disease which at the present time can be diagnosed by labora-
tory methods (White, 1918). Sixty years ago Doénhoff (1857) ob-
served an infectious condition among adult bees in which, upon
examining the stomach of affected bees, small oval bodies were
found. This work had been practically forgotten until Zander
(1909) reported some interesting findings in a disorder of adult
bees. ‘He found that the stomach wall of bees taken from colonies
suffering from what he called “malignant dysentery” contained a.
protozoan parasite. To this parasite he gave the name Nosema apis.

In England (Graham-Smith, Fantham, Porter, Bullamore, and
Malden, 1912) Nosema infection in bées has been associated with a
disorder referred to as the Isle of Wight bee disease. Recent in-
vestigations in Scotland (Anderson and Rennie, 1916) have lead to
a somewhat different view. As Nosema apis occurs in the group
Microsporidia the name “microsporidiosis” has been given to the
disease (Fantham and Porter, 1912).

Nosema disease is widely distributed. It occurs in Germany,
Australia, Switzerland, and England at least. The junior author
(White, 1914) has found the parasite Vosema apis in samples of bees
from a large number of the States of the United States and from
Canada. The disease weakens and even kills colonies and is there-
fore one of interest to beekeepers. The exact losses from it are not
known, but in America they are less than has been attributed to it in
some other countries.

OBTAINING THE BEES.

Hither dead or living bees are suitable for examination. Dead
bees may be dry and still be suitable material. Living bees for
examination can be sent very satisfactorily in mailing cages such
as are used by queen breeders; dead ones may be sent in any con-
venient way. A complete history of the colony and apiary as to
disease should accompany the bees.

GROSS CHARACTERS.

The presence of various symptoms has been mentioned as being
of importance in the diagnosis of Nosema infection. Among these
are noted the spotting of the hive with feces, abdominal distention,
the presence of shiny bees devoid of hair, and the activity of the
bees, either in the cages or when free. These are of questionable
value. It is upon the presence or absence of Nosema spores that the
diagnosis is based. Bees otherwise apparently healthy may, upon
examination of the stomach, show the presence of spores of Nosema
apis in large numbers.

14 BULLETIN 671, U. S. DEPARTMENT OF AGRICULTURE.

MICROSCOPIC FINDINGS.

The bees if alive may be killed easily by crushing the thorax be-
tween the jaws of a pair of dissecting forceps. Then the thorax is
grasped by the thumb and finger of one hand, the tip of the abdomen
is grasped with a pair of forceps held in the other, and by gentle
traction the ventriculus (stomach) and hind gut usually come away
entirely and may be teased apart for examination, or the whole gut
may be crushed under a cover glass and examined. In making a
diagnosis at least 10 bees should be examined. Spores of Nosema
apis if present are easily recognized, being oval, highly refractile
bodies (PI. II, fig. 2). Usually they occur in large numbers crowd-
ing the field. They stain with difficulty, and for diagnostic pur-
poses water mounts unstained are satisfactory. The young forms
of the parasite when present are quite difficult of detection, and
should not be depended upon in the diagnosis.

Occasionally protozoa other than Nosema apis have been en-
countered in the examination of adult bees. These have no relation
to Nosema disease, however, and may be disregarded in its diagnosis.

LITERATURE.

Much has already been written on bee diseases. The journals on
beekeeping contain numerous articles pertaining to them. Bulletin
No. 98 of the Bureau of Entomology briefly reviews a number of
papers, published prior to 1912, dealing with the causes of these
diseases. The papers reviewed and the publications cited, together
with the papers in the following list and the references which they
contain, comprise a fairly comprehensive résumé of all the literature
detailing work done on these diseases.

With regard. to further papers to appear soon, it is announced
that studies have been made on American foulbrood and European
foulbrood, similar to those on sacbrood (White, 1917) and Nosema
disease (White, 1918) and that the results are now being prepared
for publication.

ANDERSON, JOHN, and RENNIE, JOHN.
1916. Observations and experiments bearing on ‘“ Tai of Wight” disease
in hive bees. Jn Proc. Roy. Phys. Soc. Edinb., Session 1915-1916, v. 20,
pt. 1, p. 23-61, 1 pl.

ARISTOTLE. (B. C. 384-822.)

1783. Histoire des Animaux d’Aristote avec la Traduction Francoise (Notes
sur Histoire des Animaux d’Aristote) par M. Camus [Greek and French
on opposite pages]. Paris. 2yv. 4°.

Volume 1, Book IX, page 615: Bee diseases.

1910. The works of Aristotle Translated into English under the Editorship
of J. A. Smith [and] W. D. Ross. Oxford. v. 4, Historia animalium by
D’Arcy Wentworth Thompson.
Book IX, p. 626b: Bee diseases.

DIAGNOSIS OF BEE DISEASES. 15

CHESHIRE, F. R., and CHEYNE, W. W.
1885. The pathogenic history and history under cultivation of a new bacillus
(B. alvei), the cause of a disease of the hive bee hitherto known as foul
brood. Jn Jour. Roy. Micros. Soe. [London], ser. 2, v. 5, pt. 2, p. 581-601,
pl 10, 11.

DzIERZON, JOHANNES.
1882. Dzierzon’s Rational Bee Keeping; or the Theory and Practice of Dr.
Dzierzon. Translated from the latest German edition by H. Dieck and
S. Stutterd. Edited and revised by Chas. Nash Abbott. London.

DONHOFF and LEUCKART.
1857. Ueber die Fadenpilz im Darm der Biene. Jn Bienen-zeitung [Hich-
stiidt], v. 18, no. 6, p. 66-67.

FANTHAM, H. B., and PorTEer, ANNIE.
1912. The morphology and life history of Nosema apis and the significance
of its various stages in the so-called “Isle of Wight” disease in bees
(Microsporidiosis). Jn Ann. Trop. Med. and Parasit., v. 6, no. 2, p.

163-195, col. pl. 14-16.
Page 189: References.

GRAHAM-SmiTH, G. S., FANTHAM, H. B., PorTER, ANNIE, BULLAMORE, G. W.,
and MaALpEen, W.
1912. Report on the Isle of Wight bee disease (Microsporidiosis). In

Sup. 8 to the Jour. Bd. Agr. [London], v. 19, no. 2. 148 p., 5 pl.
Pages 139-143: Bibliography.

McCray, A. H.
1916. Some difficulties in gross diagnosis of the infectious brood diseases
of bees. Jn Jour. Econ. Ent., v. 9, no. 1, p. 192-196.
1917. The spore-forming bacteria of the apiary. Jn U. S. Dept. Agr. Jour.
Agr. Research, v. 8, no. 11, p. 399-420, 6 fig., pl. 938-94.

Mo.ritTor-MUHLFELD.
1868. Die Faulbrut, ibre Entstehung, Fortpflanzung und Heilung. In
Bienen-zeitung [EHichstédt], Jahrg. 24, Nro. 8, p. 938-97.
ScHIRACH, A. G.
1771. Histoire Naturelle de la Reine des Abeilles, avec l’Art de Former
des Essaims. Le Haye.
WHITE, G. F.
1907. The cause of American foul brood. U.S. Dept. Agr. Bur. Ent. Cire.
94. 4p.
1912. The cause of European foul brood. U. S. Dept. Agr. Bur. Ent.
Cire. 157. 15 p., 10 fig.
1913. Sacbrood, a disease of bees. U.S. Dept. Agr. Bur. Ent. Cire. 169.
5 p.
1914. Destruction of germs of infectious bee diseases by heating. U. S.
Dept. Agr. Bul. 92. (Contribution from the Bureau of Entomology.)
8 p.
1917. Sacbrood. U. S. Dept. Agr. Bul. 431. (Contribution from the Bu-

reau of Entomology. Professional paper.) 54 p., 33 fig., 4 pl.
Literature cited, p. 538-54.

1918. Nosema disease. U.S. Dept. Agr. Bul. —. (Contribution from the

Bureau of Entomology. Professional paper.) (In press.)
Literature cited.

ZANDER, ENOCH.
1909. Tierische Parasiten als Krankheitserreger bei der Biene. Jn Miinch-
ener Bienen-zeitung, 1909, Heft 9. 11 p., 3 fig.

ADDITIONAL COPIES

OF THIS PUBLICATION MAY BE PROCUEED FROM
THE SUPERINTENDENT OF DOCUMENTS
GOVERNMENT PRINTING OFFICE
WASHINGTON, D. C.

AT

5 CENTS PER COPY

NATIONAL AGRICULTURAL LIBRARY

WM

1022829783